hSGLTs-CHO-K1稳定转染细胞株的建立及芒果苷对SGLTs转运蛋白表达的影响  

Establishment of CHO-K1 cell lines stably expressing hSGLTs structure protein and effect of mangiferin on expression of SGLTs

作　　者：张富东[1,2,3] 高丽辉[2] 冯国华[2] 牛艳芬[2] 刘旭[2] 李玲[2] 

机构地区：[1]乐山职业技术学院,四川乐山614000 [2]昆明医科大学生物医学工程研究中心,云南昆明650500 [3]乐山老年病专科医院,四川乐山614000

出　　处：《中国药理学通报》2013年第4期553-556,共4页Chinese Pharmacological Bulletin

基　　金：云南省应用基础研究计划重点项目(No 2006C009Z)

摘　　要：目的将hSGLT1和hSGLT2基因分别转染入CHO-K1细胞,以根皮苷为阳性对照,研究芒果苷对hSGLT1-CHO-K1和hSGLT2-CHO-K1中hSGLT1及hSGLT2蛋白表达的影响。方法①脂质体介导重组hSGLT1基因和hSGLT2基因转染CHO-K1细胞,将hSGLT1和hSGLT2的编码区cDNA与真核表达载体pReceiver-M03相连接后,转染入CHO-K1细胞中,以倒置荧光显微镜观测绿色荧光发光位置。同时,以抗生素G418对转染细胞进行7 d压力筛选和96孔板单克隆,以得到稳定转染细胞系。②用Western blot探讨不同浓度芒果苷和根皮苷对hSGLT1及hSGLT2蛋白表达的影响,初步分析其作用机制。结果①脂质体介导重组hSGLT1基因和hSGLT2基因转染CHO-K1细胞48 h后,细胞表面出现明显的绿色荧光,呈环状,转让率达60%～70%,Western blot结果提示转染细胞有明显的SGLT1和SGLT2蛋白表达。②芒果苷与hSGLT1-CHO-K1和hSGLT2-CHO-K1分别作用48h后,经Western blot检测显示,芒果苷组的hSGLT1/β-actin和hSGLT2/β-actin灰度比值均低于正常组,且具有浓度依赖性,与未处理组相比,差异具有统计学意义(P<0.05);与等分子的根皮苷组相比,差异无统计学意义。结论①建立了稳定转染的hSGLT1-CHO-K1和hSGLT2-CHO-K1细胞系。②芒果苷能下调hSGLT1-CHO-K1和hSGLT2-CHO-K1细胞SGLT1和SGLT2蛋白的表达量,其效价与根皮苷相当。Aim To connect the full cDNA of hSGLT1 and hSGLT2 with the vector pReceiver-M03 and transfect them to CHO-K1 cells,and to investigate the effects of mangiferin on hSGLT1 and hSGLT2 expression in CHO-K1-SGLT1 and CHO-K1-SGLT2,compared to phloridzin as a positive control.Methods The full length cDNA of SGLT1 and SGLT2 was connected to the vector pReceiver-M03 and transfected into CHO-K1 cells by Lipofectamine2000.The expression of exogenous genes was identified by fluorescence microscope.The stable transfect cells would be obtained by screening of G418.The changes of SGLT1 and SGLT2 expression in CHO-K1-SGLT1 and CHO-K1-SGLT2 exposure to mangiferin or phloridzin were observed by the Western blot.Results Forty-eight hours after transfection,the circular green fluorescence in the cell membrane was observed under fluorescence microscope.The transfection efficiency was 60%~70%.The results of Western blot suggested that hSGLT1 and hSGLT2 expressed in CHO-K1 cells.Moreover,48 hours after CHO-K1-SGLT1 and CHO-K1-SGLT2 exposure to mangiferin or phloridzin,Western blot analysis revealed that hSGLT1/β-actin and hSGLT2/β-actin were significantly decreased compared with untreated cells（P＆lt;0.05）,but the effect of mangiferin was not significant compared to phloridzin group（P＆gt;0.05）.Conclusions The stable transfected cells have been obtained successfully.Mangiferin can inhibit the expression of SGLT1 and SGLT2,which is similar to the effect of phloridzin.

关 键 词：芒果苷 根皮苷 糖尿病 SGLT1 SGLT2 转染 WESTERN BLOT 

分 类 号：R284.1[医药卫生—中药学] R329.2[医药卫生—中医学]